Proc. Natl. Acad. Sci. USAVol. 86, pp. 4982-4986, July 1989Cell Biology

Mitosis-specific monoclonal antibody MPM-2 inhibits Xenopusoocyte maturation and depletes maturation-promoting activity

(M-phase induction/meiosis/antibody iqjection/protein phosphorylation/ceil cycle)

JIAN KUANG*, JI-YING ZHAO*, DAVID A. WRIGHTt, GRADY F. SAUNDERSt, AND POTU N. RAo*§Departments of *Medical Oncology, tMolecular Genetics, and *Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center,1515 Holcombe Boulevard, Houston, TX 77030

Communicated by David Prescott, April 13, 1989 (received for review January 26, 1989)

ABSTRACT MPM-2, a monoclonal antibody specific forcells in mitosis, recognizes a family of proteins that share acommon phosphorylated epitope. In this study we have shownthat during the maturation of Xenopus laevis oocytes inducedby progesterone, phosphorylation of MPM-2 antigens coin-cided with the appearance of MPF activity. When MPM-2(0.7-1.4 Ig per oocyte) was injected into oocytes prior toprogesterone stimulation, MPF activity failed to appear andinduction of maturation was inhibited as judged by bothgerminal-vesicle breakdown and white-spot formation. Fur-ther, MPM-2 was able to neutralize as well as immunodepleteMPF activity from mitotic HeLa cell and mature oocyteextracts. These results suggest that MPM-2 recognizes eitherMPF itself or a protein(s) that regulates MPF activity and thatthe kinase that phosphorylates MPM-2 antigens may be a keycomponent in the regulation of M-phase induction.

The regulation of the initiation of the M phase (mitosis andmeiosis) of the eukaryotic cell cycle has been studied inten-sively (reviewed in ref. 1). In particular, the identification andcharacterization of the maturation-promoting factor or M-phase-promoting factor (MPF) have drawn much attention(1). MPF is an activity present in M-phase cells of all speciestested that upon microinjectiondinduces immature oocytes toundergo meiotic division (maturation). This activity oscil-lates during the cell cycle and always correlates with theinitiation and maintenance of the M phase (2). Therefore,MPF has been considered as the universal trigger of theinterphase/M-phase transition.MPF has been purified 3500-fold from mature oocytes of

Xenopus (3). The purified MPF consists oftwo major proteinsof32 kDa and 45 kDa and exhibits protein kinase activity. The32-kDa component of MPF has been demonstrated by im-munocrossreactivity to be the Xenopus homologue of thecdc2 gene product of Schizosaccharomyces pombe (4). Dun-phy et al. (5) reached the same conclusion by using affinitydepletion of the cdc2 gene product. In both studies, the45-kDa protein was associated with the 32-kDa protein. Thus,MPF is considered to be a protein complex expressing cdc2kinase activity.

In Xenopus oocytes and in rapidly cleaving embryos, MPFis stored in a latent form during interphase (6-8) and isactivated during the induction ofM phase. In vivo activationofMPF requires protein synthesis both for oocyte maturationand for interphase/mitosis transition in rapidly cleavingembryos (6, 9), suggesting that the newly synthesized pro-tein(s) might act as a positive regulator of MPF. The devel-opment ofan in vitro system capable of activating latent MPFhas led to the discovery of an inhibitor of MPF activationtermed INH (8). Therefore, MPF is under both positive andnegative control.

Activation of MPF always correlates with a high level ofprotein phosphorylation (10-16). However, the exact roles ofthe M-phase-specific phosphorylations are not known. In thisstudy, we have characterized the mitosis-specific phospho-rylation detected by a mitosis-specific monoclonal antibody,MPM-2. MPM-2, an IgG antibody, reacts specifically withmitotic cells of different species by indirect immunofluores-cence (17) and on immunoblots recognizes a family of phos-phoproteins. When MPM-2 antigens are dephosphorylated invitro, their antigenicity is lost (17). Thus, MPM-2 detectsmitosis-specific phosphorylation ofa group ofproteins duringthe G2/M transition. In this study, we have found that duringprogesterone-induced oocyte maturation, phosphorylation ofMPM-2 antigens coincides with the appearance of MPFactivity. MPM-2 inhibits oocyte maturation upon microin-jection and neutralizes and immunodepletes MPF activityfrom M-phase extracts. These results indicate that MPM-2recognizes a mitosis-specific phosphorylation of either MPFitself or a protein(s) that regulates MPF activity.

MATERIALS AND METHODSMaterials. Frogs (Xenopus laevis) were obtained from

Nasco (Fort Atkinson, WI). Affi-Gel blue (100-200 mesh),Bio-Gel A-1.5m, alkaline phosphatase-conjugated goat anti-mouse IgG, and protein A MAPS II kit were from Bio-Rad.Centricon-30 concentrators were from Amicon. Most otherreagents were from Sigma.

Purification of Monoclonal Antibodies. Three successivechromatographies were performed to purify antibodies frommouse hybridoma ascites. (i) The IgG fraction from theascites was purified by protein A-mediated affinity chroma-tography using the Affi-Prep protein A MAPS II system. (ii)The eluate, containing mostly IgG, was subjected to gelfiltration through a Bio-Gel A-1.5m column (1.5 x 46 cm)preequilibrated in Tris-buffered saline (TBS: 10 mMTris-HCI/150mM NaCl, pH 7.5). (iii) The IgG fractions fromgel filtration were pooled, concentrated with Centricon-30,and subjected to HPLC on hydroxyapatite. Then the IgGfractions were pooled again and concentrated to 20 mg/ml inTBS with Centricon-30. The concentrated antibodies werestored at -20'C until used. MPM-2 and the control antibody(MPM-7) were purified in the same manner.Assay for Inhibition of Oocyte Maturation by Antibodies.

Purified antibodies (20 mg/ml) were diluted serially with TBSbefore injection. Seventy nanoliters of diluted antibodies orTBS was injected into at least 10 oocytes just prior to theexposure of oocytes to progesterone (10 ,ug/ml). When allTBS-injected oocytes had matured, as judged by white-spot

Abbreviations: MPF, maturation-promoting factor; GVBD, germi-nal-vesicle breakdown; WSF, white-spot formation; MFD, maximalfold dilution of MPF activity that causes maturation in 50% ofinjected oocytes; EB, extraction buffer; TBS, Tris-buffered saline.§To whom reprint requests should be addressed.

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formation (WSF), antibody-injected oocytes were scored forboth WSF and germinal-vesicle breakdown (GVBD) afterdissection. These two events usually coincide in the normalmaturation process, although GVBD can be induced withoutWSF under certain circumstances.

Preparation of Oocyte Extracts. For small-scale preparationof oocyte extracts (50 oocytes) in the time-course studies,oocytes were rinsed twice in cold extraction buffer (EB: 80mM sodium (3-glycerophosphate/20 mM EGTA/15 mMMgCl2/1 mM dithiothreitol/1 mM ATP/1 mM phenylmeth-ylsulfonyl fluoride, pH 7.4; ref. 18) and transferred intoEppendorf tubes (500 pl). After excess buffer was removedwith a gentle spin, an equal volume of 2x EB containing amixture of protease inhibitors [leupeptin, 5 Jug/ml; L-7-amino-1-chloro-3-tosylamido-2-heptanone ("Na-tosyl-L-lysine chloromethyl ketone"), 0.1 mM; phenylmethylsul-fonyl fluoride, 1 mM; N-ethylmaleimide, 0.5 mM; a2-macroglobulin, 1 ,g/ml] was added. The oocytes werehomogenized by pipetting and the homogenates were micro-centrifuged at 15,000 x g for 10 min at 40C. The materialbetween the pellet and the lipid cap was recovered. Analiquot was diluted immediately with 3 x NaDodSO4/PAGEsample buffer. Microinjection of the extract into oocytes wasdone within 20 min to assay for MPF activity. For large-scalepreparation of mature oocyte extracts, the unfertilized eggswere dejellied in 2% cysteine (pH 7.9). After three washes in100mM NaCl, the eggs were rinsed in cold EB. The eggs werecrushed by centrifugation at 15,000 x g for 25 min and thecytoplasmic material was recovered. Pre-MPF was preparedfrom immature oocyte extract (22).

Preparation of Mitotic HeLa Cell Extract. HeLa cells weresynchronized in mitosis as described (19). The mitotic cellextract was prepared by pelleting the cells at 600 x g for 5 minat 4°C, removing the excess medium, and suspending thecells at 4 x 107 per ml in 2x EB containing a mixture ofprotease inhibitors. Cells were disrupted by sonication andthe lysates were centrifuged at 100,000 x g for 60 min at 4°Cin a Beckman L5-50 ultracentrifuge. The supernatants werestored at -70°C until used. Because the extracts made in 1 xEB and 2x EB showed similar MPF activity on microinjec-tion, we chose to use 2x EB, as the extracts would be dilutedwith an equal volume of TBS in neutralization and immun-odepletion studies.Assay for MPF Activity. Cell extracts were assayed for the

ability to induce oocyte maturation upon microinjection andquantitated by the maximal fold dilution (MFD) that causedmaturation in 50% of the injected oocytes. Samples werediluted serially with EB just prior to microinjection, and 70 nlof diluted sample was injected into each oocyte. At least 10oocytes were injected with each sample. The injected oocyteswere incubated for 2 hr in Barth's solution (20) at 22°C andscored for maturation by WSF at the animal pole and theabsence of germinal vesicles after fixation in 5% (wt/vol)trichloroacetic acid and dissection. The MFD for maturationinduction in 50% of oocytes was determined by plotting theinverse of the dilution against the percentage of oocytesshowing GVBD.

Neutralization and Immunodepletion of MPF. In neutral-ization studies, purified antibodies (20 mg/ml) were dilutedserially with TBS. The diluted antibodies were mixed with anequal volume of mitotic HeLa cell extract or meiotic Xenopusoocyte extract. After the mixture was incubated on ice for 2hr, 70 nl was injected into each oocyte. Two hours later, theoocytes were fixed with 5% trichloroacetic acid and dissectedto determine GVBD.For immunodepletion studies, IgG from ascites was im-

mobilized to protein A-conjugated beads (protein A-agaroseor Affi-Prep protein A matrix) by using the MAPS II kit. Theimmunoaffinity beads were equilibrated and washed withTBS and then mixed with an equal volume of mitotic HeLa

cell extract or meiotic Xenopus oocyte extract. After themixture was rotated for 3-4 hr at 40C, the beads were pelletedand the supernatants were assayed for MPF activity.NaDodSO4/PAGE and Immunoblot Analysis. Proteins

were separated by NaDodSO4/8% PAGE (21), electrophoret-ically transferred onto nitrocellulose (22), and stained withMPM-2 by using alkaline phosphatase-conjugated goat anti-mouse IgG as the second antibody (23).

RESULTSAppearance of MPM-2 Antigens Coincides with the Appear-

ance of MPF Activity During Oocyte Maturation. MPM-2antigens are present inM phase and absent in interphase (17).To define more precisely when MPM-2 antigens appearedduring the G2/M transition, we monitored the appearance ofthe expression of MPM-2 antigens in relation to the appear-ance of MPF activity during the progesterone-induced mat-uration ofXenopus oocytes. Immature oocytes had few or noMPM-2 antigens (Fig. 1A). Some MPM-2 antigens of about180 kDa began to appear at 50 min after the exposure ofoocytes to progesterone, when MPF activity was still unde-tectable. By 150 min, when MPF activity was detectable,MPM-2 antigens were more numerous and were expressed atmuch higher levels. The immunoblot showed that two of theantigens (180 and 58 kDa) were the most abundant. At 250min (i.e., between the first and second meiotic divisions),there was a significant decrease in MPF activity. This wasassociated with a noticeable decrease in the 58-kDa antigen.These results indicate that, in general, appearance of MPM-2antigens correlates well with MPF activity during oocytematuration, although expression of some MPM-2 antigens ata low level preceded the detection of MPF activity.

Protein Synthesis Is Required for the Appearance of MPM-2Antigens in Progesterone-Stimulated Oocytes. During proges-terone-stimulated maturation, protein synthesis is required

A

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%GVBD

MPF (MFD)

2 3 4 5 6 7 8

0 50 100 150 200 250 300

0 0 0 10 95 100 1000 0 0 4 8 3 8

B

9 10 11 12

9 10 11 12

50 100 150 200

0 0 a aa a a 0

FIG. 1. Expression of MPM-2 antigens and the appearance ofMPF activity during progesterone-induced oocyte maturation. De-folliculated X. laevis oocytes were incubated in Barth's solution inthe absence (A) or presence (B) of cycloheximide (100 Aug/ml).Progesterone was added to the medium at time zero. Every 50 minthereafter, oocytes were scored for WSF and GVBD, and a batch ofoocytes was taken to make extracts. The extracts were analyzed forMPF activity by microinjection into immature oocytes and for theexpression of MPM-2 antigens on immunoblots. Proteins from theextracts were separated by NaDodSO4/8% PAGE, electrophoreti-cally transferred to nitrocellulose sheets, and stained with MPM-2.The time of sampling, percent GVBD, and relative MPF activity(expressed as the MFD) are indicated below each lane. In cyclohex-imide-treated oocytes, neither MPM-2 antigens nor MPF activity wasdetectable (B). In oocytes not treated with cycloheximide (A),MPM-2 antigens of about 180 kDa appeared as early as 50 min afterprogesterone stimulation, whereas MPF activity was not detectableuntil 150 min. A 58-kDa band appeared with the detection of MPFactivity. Lane 1 shows protein size standards.

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for the appearance of MPF activity. To test whether proteinsynthesis is necessary for the expression ofMPM-2 antigens,cyclohexirnide (100 ,ug/ml) was added to the culture medium30 min before the exposure ofoocytes to progesterone. Whenprotein synthesis was inhibited, neither MPF activity norMPM-2 antigens could be detected (Fig. 1B). These resultssuggest that appearance of MPM-2 antigens is an eventdownstream of the nascent protein synthesis required foroocyte maturation.Appearance of MPM-2 Antigens Is Due to Phosphorylation

of Preexisting Proteins. In HeLa cells, the proteins ofMPM-2antigens are synthesized in interphase and phosphorylatedduring mitosis induction (31). Since MPM-2 recognizes onlythe phosphorylated form of these proteins, the appearance ofMPM-2 antigens in mitosis indicates their phosphorylation.To determine whether the appearance of MPM-2 reactivityduring oocyte maturation represents phosphorylation of pre-existing proteins, we analyzed the expression of MPM-2antigens during MPF-induced maturation as well as activa-tion of pre-MPF in vitro.MPF can induce oocyte maturation by activating the

endogenous pool of inactive MPF. During MPF-inducedmaturation, protein synthesis is not necessary for the induc-tion of the first meiotic division but is required before theoocytes can progress into the second meiotic metaphase (8).We tested whether MPM-2 antigens appeared in MPF-injected oocytes in the absence of protein synthesis. Seventynanoliters of mitotic HeLa cell extract (MFD = 3) wasinjected into oocytes with or without the inhibition of proteinsynthesis by cycloheximide (100 ,ug/ml). At intervals duringthe next 200 min, extracts were made and assayed for MPFactivity and the expression of MPM-2 antigens (Fig. 2A). Asexpected (8), cycloheximide did not prevent the appearanceof high MPF activity in the first meiotic cycle. However, inthe absence of protein synthesis, the MPF-injected oocytescould not progress into the second meiotic metaphase, asindicated by a drop in MPF activity at 200 min. MPM-2antigens appeared at similar levels with or without proteinsynthesis but were greatly decreased in the cycloheximide-treated oocytes at 200 min (Fig. 2A).

Cyert and Kirschner (22) have shown that the pre-MPFfraction (the 0-33% ammonium sulfate fraction of the high-speed immature oocyte extract) rapidly generates MPF ac-tivity without the addition of active MPF. To test whetherMPM-2 reactivity appeared during the activation of pre-MPFin this cell-free system, pre-MPF was incubated at room tem-perature in the presence or absence of an ATP-regener-ating system, which is required for activation of pre-MPF. Inthe absence of the ATP-regenerating system, neither MPFactivity nor MPM-2 reactivity was detectable (Fig. 2B). Incontrast, both MPF activity and MPM-2 antigens appearedwhen the ATP-regenerating system was present.Thus, the expression of MPM-2 antigens correlates with

the presence of MPF activity, as shown in progesterone-stimulated oocyte maturation. Further, since protein synthe-sis is not necessary for either MPF-induced maturatipn oractivation ci rre-MPF in vitro, we conclude that the appear-ance of Ml -2 antigens during oocyte maturation must bedue to the sphorylation of preexisting proteins.MPM-2 . .its GVBD and WSF upon Microinjection. The

appearance of MPF activity concurrently with the appear-ance ofMPM-2 antigens (i.e., the phosphorylation of MPM-2antigens) suggests that the phosphorylation detected byMPM-2 might be involved in the induction of M phase.Therefore, we tested whether MPM-2, upon microinjection,could inhibit progesterone-stimulated oocyte maturation. Weused another mitosis-specific monoclonal antibody (MPM-7)as a control. Both MPM-2 and MPM-7 were purified bysuccessive protein A affinity chromatography, gel filtration,and hydroxyapatite HPLC. Oocytes were injected with an-

A

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MPF (MFD) -

i

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0 60 80 90

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FIG. 2. (A) Appearance of MPM-2 antigens and MPF activity inoocytes injected with mitotic HeLa cell extract in the presence orabsence of protein synthesis. Defolliculated X. laevis oocytes wereincubated in Barth's solution in the presence (lanes 6-9) or absence(lanes 2-5) of cycloheximide (100 pg/ml) for 50 min. Seventynanoliters of mitotic HeLa cell extract (MFD = 3) was injected intooocytes at time zero. At 100, 150, and 200 min, oocytes were scoredfor GVBD as judged by WSF. Fifty oocytes were taken for eachtreatment at each time point to make extracts. The extracts were thenassayed for MPF activity by microinjection into immature oocytesupon serial dilution. The expression ofMPM-2 antigens was analyzedon immunoblots stained with MPM-2 antibody. For time zero (lanes2 and 6) the oocyte extracts were mixed with corresponding amountsof mitotic HeLa cell extract (70 nl per oocyte). (B) Appearance ofMPM-2 antigens during in vitro activation of pre-MPF. The 0-33%ammonium sulfate fraction of a high-speed extract of immatureoocytes was incubated at room temperature for 30 min in thepresence (lane 3) or absence (lane 2) of an ATP-regenerating system(1 mM ATP, 50 ,g of creatine kinase per ml, and 10 mM creatinephosphate). Both samples were then assayed for MPF activity bymicroinjection into immature oocytes and for MPM-2 reactivity byimmunoblot analysis.

tibodies or buffer just before they were exposed to proges-terone. MPM-2-injected oocytes were scored after the buffer-or MPM-7-injected oocytes matured, as judged by WSF andGVBD. MPM-2 inhibited both WSF and GVBD in a dose-dependent manner (Fig. 3). In all experiments, 70 nl of theantibody was injected into each oocyte. Inhibition ofGVBDrequired higher concentration ofthe antibody (10-20 mg/ml),and the inhibition was not complete (80%). In contrast,complete inhibition of WSF occurred at a much lower con-centration (0.4 mg/ml). When the incubation of MPM-2-injected oocytes was continued after all the control oocyteshad matured, the percentage of the oocytes that underwentGVBD sometimes increased, ranging from 0% to 50%, indi-cating that in certain cases MPM-2 caused a delay in oocytematuration rather than a complete block. We also observedsome variability among oocytes from different frogs withregard to the percentage of inhibition and the lowest dosesneeded for inhibition (Table 1). Nevertheless, MPM-2 con-sistently showed an inhibitory effect on oocyte maturation.MPM-2 Inhibits the Appearance of MPF Activity in Proges-

terone-Stimulated Oocytes. Oocytes were injected with MPM-2 before they were exposed to progesterone. The appearanceof MPF activity and the expression of MPM-2 antigens werethen analyzed in MPM-2- and buffer-injected oocytes. MPFactivity was not detectable in MPM-2-injected oocytes at atime when buffer-injected oocytes showed high MPF activity(Fig. 4). This indicates that at least one MPM-2 antigen isneeded for the appearance of MPF activity. Furthermore,expression of MPM-2 antigens was also greatly decreased inMPM-2-injected oocytes, with the 58-kDa antigen beingcompletely absent and the 180-kDa antigen being expressedat a much reduced level. This suggests that the MPM-2

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FIG. 3. Effect of MPM-2 on WSF and GVBD in progesterone-stimulated oocytes. MPM-2 at various concentrations (70 nl) wasinjected into batches of 20 oocytes, which were then immediatelyexposed to progesterone. When all the control oocytes injected withTBS or MPM-7 exhibited white spots, the MPM-2-injected oocyteswere scored for WSF, fixed, and dissected for GVBD scoring.Oocytes injected with TBS or MPM-7 at corresponding concentra-tions matured normally.

antigen involved in the appearance of MPF activity may inturn affect the phosphorylation of other MPM-2 antigens.MPM-2 Neutralizes and Immunodepletes MPF Activity. The

most plausible explanation for our results is that one of theMPM-2 antigens might be MPF itself or a protein required toactivate MPF. Therefore, we tested the ability of MPM-2 toneutralize MPF upon mixing and to immunodeplete MPFactivity from M-phase cell extracts. In neutralization studies,meiotic extracts from Xenopus oocytes or mitotic extractsfrom HeLa cells were mixed in vitro with MPM-2 or thecontrol antibody MPM-7 (final concentration of antibodies, 5mg/ml). After a 2-hr incubation at 0C, the samples wereassayed for MPF activity with or without dilution with anequal volume of EB. The concentrations of the unboundantibodies in the mixture were <5 mg/ml without dilution or2.5 mg/ml with dilution, which were not high enough toinhibit the induction ofGVBD in 50%o ofprogesterone-treatedoocytes. Whereas M-phase extracts mixed with MPM-7 orTBS were able to induce GVBD even upon dilution, theMPM-2-mixed extracts did not induce GVBD with or withoutdilution (Table 2). The results indicate that MPF activity wasneutralized by MPM-2 but not by MPM-7 or TBS. To excludethe possibility that the failure to detect MPF activity inMPM-2-mixed M-phase extracts was attributable to the pres-ence of unbound antibody instead of the inactivation ofMPFin M-phase extracts, immunodepletion studies in which an-

Table 1. MPM-2 inhibits progesterone-induced maturation ofXenopus oocytes

Experi- GVBD50,* % GVBDment hr MPM-2 MPM-7

1 7.0 10 1002 6.0 20 1003 3.0 20 1004 3.0 30 1005 4.0 10 1006 6.0 0 1007 3.0 30 1008 3.0 20 100

In all the experiments 70 nl of the antibody (MPM-2 or MPM-7) at20 mg/ml was injected into each oocyte. MPM-2-injected oocyteswere scored for GVBD when MPM-7-injected oocytes had matured.*Time required for 50% of the control (MPM-7-injected) oocytes tomature after exposure to progesterone. This time varies amongoocytes from different frogs.

180116 -

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LANE No.

TIME (MIN)

SGVBD

MPF(MFD)

2 3 4 5 6

0 50 100 150 200

O O 0 50 100

o 3 4 2

B

7 8 9 10 11 12

8 9 10 11 12

0 50 100 150 200

0 0 000

0 0 000

FIG. 4. Effect of MPM-2 on the appearance of MPF activity andMPM-2 antigen expression in progesterone-stimulated oocytes. Xe-nopus oocytes were injected with 70 nl ofMPM-2 (20 mg/ml) or TBSand then immediately exposed to progesterone (10 ,g/ml). Time-course studies were performed as described in the legend to Fig. 1.The time of sampling, percent GVBD, and MPF activity (MFD) areindicated below each lane. (A) Oocytes injected with TBS. (B)Oocytes injected with MPM-2. The dark band indicated by an arrowin B is the heavy chain of the injected antibody.

tibodies were immobilized to protein A beads were per-formed. In addition to MPM-7, mouse IgG or RDA-1 (nucle-olus-specific monoclonal antibody) was also used as a con-trol. The immunoaffinity beads were mixed with an equalvolume of M-phase extract. After mixing at 4°C for 3 hr, thebeads were pelleted by centrifugation and the supernatantswere assayed for MPF activity. Only MPM-2 was able todeplete MPF activity from M-phase extracts (Table 3). Whenthe depleted extracts were analyzed on immunoblots, theMPM-2-depleted extracts showed very little of the MPM-2antigens, in contrast to the extracts depleted by controlantibodies (Fig. 5). Some bands disappeared completely andothers were barely perceptible. These results indicate thatMPM-2 can deplete MPF activity from both mitotic andmeiotic cell extracts.

DISCUSSIONMPM-2, a mitosis-specific monoclonal antibody raised in ourlaboratory, recognizes a family of phosphoproteins in mitoticcells (17). In HeLa cells, these proteins are synthesized ininterphase and phosphorylated in M phase. The antibodyrecognizes only their phosphorylated form inM phase. In thisstudy, we have attempted to define the role that the phos-phorylation ofMPM-2 antigens plays in the M-phase inductionpathway. We have shown that during progesterone-stimulatedoocyte maturation, there is a general correlation between theexpression of MPM-2 antigens and the appearance of MPFactivity, although expression ofsome MPM-2 antigens at a lowlevel preceded the detection of MPF activity. When protein

Table 2. Neutralization of MPF activity by in vitro mixing ofMPM-2 with mitotic HeLa cell extract or mature oocyte extract

Oocytes showing GVBD, no./no. injectedMitotic HeLa extract Mature oocyte extract

Antibody Undiluted Diluted Undiluted DilutedMPM-2 0/10 0//10 0/10 0/10MPM-7 10/10 7/10 10/10 5/10None (TBS) 10/10 7/10 10/10 3/10

Five microliters of the extract (10 mg/ml) was mixed with 5 ,Al ofantibody (MPM-7 or MPM-2, 10 mg/ml) or TBS in an Eppendorftube. The mixture was incubated on ice for 3 hr (HeLa cell extract)or 2 hr (oocyte extract) and then injected, with or without 1:1 dilutionwith EB, and scored for GVBD 2 hr after injection.

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Table 3. MPM-2 immunodepletion of MPF activity from mitoticHeLa cell and mature oocyte extracts

Oocytes showing GVBD, no./no. injected

Mature oocyteMitotic HeLa extract extract

Antibody 100%O 50%0 25% 100%0 50%oMPM-2 2/10 0/10 0/10 1/10 0/10

0/10 0/10 0/10 0/10 0/10MPM-7 10/10 10/10 0/10 10/10 1/10

10/10 10/10 10/10 10/10 2/10RDA-1 10/10 10/10 0/10 10/10 2/10Mouse IgG 10/10 10/10 0/10 10/10 3/10Immunodepletion was performed as described in Materials and

Methods. The depleted extracts were assayed for MPF activitywithout dilution (100%o) or after dilution with 1 volume (50%o) or 3volumes (25%) of EB. Oocytes were scored for GVBD 2 hr afterinjection. The results are from two representative experiments.

synthesis was inhibited by cycloheximide, MPM-2 antigensdid not appear and MPF activity was not detected. Theappearance of MPM-2 antigens coincided with activation ofMPF in MPF-injected oocytes as well as in in vitro activationof pre-MPF. In neither case was protein synthesis required.Since MPM-2 only recognizes a phosphorylated epitope (31),we conclude that the appearance ofMPM-2 antigens was dueto the phosphorylation ofproteins already present in immatureoocytes. These results suggest that the phosphorylation ofMPM-2 antigens is an event downstream ofthe nascent proteinsynthesis required for progesterone-stimulated maturation andcoincides with the activation of MPF.MPM-2 neutralized and immunodepleted MPF activity

from M-phase cell extracts. These results suggest that theMPM-2 antigen involved in the appearance of MPF activitymight be MPF itselfor a regulator ofMPF present in M-phaseextracts. If MPM-2 could recognize MPF itself, then it couldinactivate MPF directly. However, if MPM-2 recognizes aregulatory protein that is necessary to keep MPF in its activeform, it could inactivate MPF indirectly. Consequently, theinactivation of either MPF or the regulator of MPF wouldgive the same result-the loss of MPF activity. Thus, weconclude that MPM-2 recognizes eitherMPF or a regulator ofMPF. Further studies would be necessary to distinguishbetween these two possibilities.

In earlier studies (24), MPM-2 was unable to inhibit theinduction ofGVBD and to immunodeplete MPF activity. Webelieve that this was due to the low levels of MPM-2 used in

kDa 1 2 3 4 5 6 7

180 nj l116 "- *-84I58 2146.5- H

36.526.6

_- ~ _ L

FIG. 5. Immunoblot of mitotic HeLa cell extracts immunode-pleted with MPM-2 or control antibodies and stained with MPM-2.Mitotic HeLa cell extract (100 ,ul) was mixed with an equal volumeofprotein A beads to which antibody was bound. After rotating at 4°Cfor 3 hr, the beads were pelleted and the supernatant was assayed forMPF activity. Thirty-three microliters of each supernatant wasmixed with 3x NaDodSO4/PAGE sample buffer for immunoblotanalysis with MPM-2. Lanes: 1, molecular mass markers; 2, nucle-olus-specific monoclonal antibody RDA-1; 3, MPM-2; 4, MPM-7; 5,mouse IgG; 6, MPM-2; and 7, MPM-7. Arrows indicate the heavy (H)and light (L) chains of IgG, which are probably due to trace amountsof immunoaffinity beads in the supernatant.

those experiments. In the present work, the inhibition ofGVBD by MPM-2 required very high doses, i.e., 70 nl of theantibody at 10-20 mg/ml. These doses are 5-10 times higherthan the doses used in the earlier studies (70 nl of 2 mg/ml).We also noticed that the amount of MPM-2 was critical forimmunodepletion of MPF activity. If the amount of antibodywas only enough to remove a portion of the antigens, thereduction in MPF activity was less than that of the MPM-2antigens. When the depleted extracts were analyzed onimmunoblots, some MPM-2 antigens disappeared completelywhile others were slightly depleted. These results indicatethat different MPM-2 antigens may have different bindingaffinities for the antibody. It is possible that in the earlierstudies, although MPM-2 was able to deplete a significantportion of its antigens from mitotic cell extracts, the antigeninvolved in MPF activity was still mainly free.

It has been proposed that MPF is activated by proteinphosphorylation (8). However, the specific phosphorylationthat is responsible for the activation of MPF has not beenidentified. This study has raised an interesting possibility:that the phosphorylation detected by MPM-2 might be re-sponsible for the activation of either MPF or a regulator ofMPF. If this is the case, the kinase that phosphorylatesMPM-2 antigens should be one of the key components in theregulation of M-phase induction.

The experiments involving in vitro activation of pre-MPF wereconducted in Dr. Marc Kirschner's laboratory with the help of TinaLee. We thank Drs. Laurence Etkin, Ramesh Adlakha, and WilliamKlein for reading the manuscript and making useful comments. Thisstudy was supported in part by Research Grants CA34783 andCA27544 (to P.N.R.) from the National Cancer Institute. J.K. issupported by a Rosalie Hites Fellowship.

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